The nuclear‐coded chloroplast 22‐kDa heat‐shock protein of <i>Chlamydomonas</i>

Abstract

A cDNA clone, pCHS62, was isolated using poly(A)‐rich RNA from heat‐shocked Chlamydomonas reinhardtii cells. The clone has a length of 1.1 kb and codes for the complete heat‐shock protein which was reported to be associated with the grana region of the thylakoid membranes and ascribes protection against photoinhibition during heat‐shock. An expression vector prepared in the pUC19 plasmid was used to obtain a fusion protein against which rabbit polyclonal antibodies have been raised. The antibodies react specifically with the heat‐shock protein of 22 kDa synthesized in vivo during heat‐shock, which is localized in the grana thylakoids, with the in vitro translated product using poly(A)‐rich RNA from heat‐treated cells as well as with the hybrid release translation product of the pCHS62 clone. The clone was sequenced. It contains a 5′ region consisting of 85 nucleotides, an open reading frame of 471 nucleotides and a non‐coding 3′ region of 600 nucleotides. Northern hybridization indicates a lenght of 1.7 kb for the messenger RNA of heat‐shock protein 22. Analysis of similarity between the derived amino acid sequence of this protein and other heat‐shock proteins demonstrates that this protein belongs to the small‐molecular‐mass plant heat‐shock protein family and also shows similarities with animal heat‐shock proteins including the presence of a short region possesing similarity with bovine α‐crystalline as reported for other heat‐shock proteins. The molecular mass of the protein as determined from the sequence is 16.8 kDa. Despite its localization in the chloroplast membranes, it does not seem to include a transit peptide sequence, in agreement with previous data. The sequence contains only a short hydrophobic region compatible with its previously reported localization as a thylakoid extrinsic protein.